The long-term goal of this project is to determine the molecular and cellular mechanisms by which testosterone regulates spermatogenesis. Testosterone is essential for fertility and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors and provide an environment required for germ cell survival and development. Numerous critical Sertoli cell activities have been found to be dependent on testosterone including 1) the formation of tight junctions between Sertoli cells that form the essential blood-testis barrier, 2) the remodeling of Sertoli cell-round spermatid attachments and the retention of round spermatids, 3) the release of mature spermatozoa. Although testosterone had been known to be required for these functions, the mechanisms by which testosterone acts are not well understood. We recently characterized an alternative, rapid (<1 min) and sustained mechanism of testosterone action (the non-classical pathway) that causes the phosphorylation and activation of the Src and ERK kinases, the epidermal growth factor receptor and the CREB transcription factor. We will test the overall hypothesis that critical spermatogenesis supporting functions of testosterone in vivo are mediated via the non-classical pathway. Aim 1 is to determine whether non-classical signaling by testosterone regulates Sertoli-Sertoli and Sertoli-germ cell adhesion in culture. We will test the hypotheses that non-classical testosterone actions are required for the formation of Sertoli-Sertoli tight junctions and that non-classical signaling is required for Sertoli-germ cell adhesion. Aim 2 is to determine whether non-classical testosterone actions regulate Sertoli-germ cell interactions in seminiferous tubules. We will test the hypothesis that non-classical testosterone actions activate kinases in seminiferous tubules and cause the release of mature spermatozoa. Aim 3 is to determine whether the non-classical pathway is required to activate Src and/or ERK kinase and downstream targets required to maintain fertility. We will test the hypothesis that testosterone activates Src and ERK kinases in rat testes in vivo and that over expression of the kinases is sufficient to release mature spermatozoa. This study will identify the mechanisms by which testosterone supports spermatogenesis and assist in divining methods to regulate male fertility.